Target-specific cytotoxic liposomes

ABSTRACT

The A fragment of the diphtheria toxin (DTA) was encapsulated in pH-sensitive liposomes. This novel reagent is extremely cytotoxic to cells expressing surface antigen which is recognized by the immunoliposome. The reagent is not toxic to cells which do not express the antigen. Thus, this reagent, or others similarly prepared represent potential anticancer reagents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of copending application Ser.No. 602,177, filed 19 Apr. 1984, now allowed U.S. Pat. No. 4,789,633 thedisclosure of which, to the extent necessary, is hereby incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Liposomes composed of phosphatidylethanolamine and oleic acid (PE/OA)become unstable and fusion-active at the weakly acidic pH of from about5 to about 6.5. These pH-sensitive liposomes can be coated with fattyacid-derivatized antibody to enhance the cytoplasmic delivery ofencapsulated molecules to antigen expressing cells.

Cytoplasmic delivery is thought to be achieved through receptor-mediatedendocytosis of the immunoliposomes. The liposomes then encounter theacidic pH of the endosome and are thought to fuse with the endosomemembrane, thus releasing the encapsulated contents into the cytoplasm.

SUMMARY OF THE INVENTION

The A fragment of the diphtheria toxin (DTA) has been encapsulated intopH-sensitive liposomes. This novel reagent is extremely cytotoxic tocells expressing surface antigen which is recognized by theimmunoliposome. The reagent is not toxic to cells which do not expressthe antigen. Thus, this reagent, or others similarly prepared, representpotential anticancer reagents.

The present invention is also directed to the use of the fragment A ofdiphtheria toxin (DTA) as a marker for cytoplasmic delivery.

The pH-sensitive immunoliposomes of the present invention are able toovercome the translocation block in DT-resistant L929 cells. It isbelieved that these liposomes are able to release DTA from an acidiccellular compartment by fusion with the endosome membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the inhibition of protein synthesis by free orliposome encapsulated toxins. Group (A) represents PE/OA immunoliposomescontaining DTA; Group (B) represents PE/OA liposomes containing DTA;Group (C) empty PE/OA immunoliposomes; Group (D) PC Immunoliposomescontaining DTA; Group (E) PC liposomes containing DTA; Group (F) DTalone; and Group (G) represents DTA alone.

FIG. 2 illustrates the blocking of cytotoxicity mediated by DTAencapsulated in pH-sensitive immunoliposomes. The data presented are;I-PE/OA (DTA): pH-sensitive immunoliposomes containing DTA; and E/OA(empty): pH-sensitive immunoliposomes not containing DTA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is known that diphtheria toxin (DT) resistant mouse cells bind andinternalize DT normally (Keen, J. H., et al., Proc. Natl. Acad. Sci.USA, 79: 2912-2916 (1982) and L929 cells possess a DT-sensitiveelongation factor 2, see for example, Guillemot, J. C., et al., CellPhys., 122: 193-199 (1985); Nakanishi, M., et al., Exp. Cell. Res., 159:399-409 (1985); Uchida, T., et al., Cell Biol., 80: 10-20 (1979) andYamazumi, M., et al., Cell. 15: 245-250 (1978)). Thus, DT resistance inL929 cells seems to result from a block in the translocation of fragmentA from the endosome into the cytoplasm.

DTA irreversibly inhibits protein synthesis in eukaryotes byenzymatically inactivating elongation factor 2, (Pappenheimer, A. M.,Jr., Ann. Rev. Biochem., 46: 69-94 (1977)). DTA is non-toxic to eitheranimals or cultured cells because it cannot reach the cytoplasm (Keen etal., supra: Pappenheimer, supra; Gill, D. M., et al., J. Biol. Chem.,246: 1492-1495 (1971) and Gill, D. M., et al., J. Biol. Chem., 246:1485-1491 (1971)).

It has been shown, however, that DTA is toxic if introduced into thecell by artificial means (Guillemot et al., supra: Nakanishi et al.,supra; Uchida et al., supra: Yamazumi et al., supra).

In accordance with the findings of other investigators, incubation ofL929 cells with unencapsulated DTA or DT had no effect on proteinsynthesis as measured by [3H]-leucine incorporated into newlysynthesized proteins. (FIG. 1).

We encapsulated DTA in liposomes prepared by a modification of thedehydration-rehydration method, Kirby, C., et al., Biotechnology, 1:979-984 (1984). This method was chosen since it does not requireexposure of encapsulated contents of either sonication or organicsolvents as do other methods of liposome preparation. Furthermore,incorporation of palmitoyl antibody (Huang, L., et al., in LiposomeTechnology, Vol. 3 79-108. CRC Press, Boca Raton, Fla. (1983)) could bereadily achieved by including the antibody in the rehydration mixture.We have used a mouse monoclonal antibody to the major histocompatabilityantigen H-2K (k) to prepare the immunoliposomes since DTA resistantmouse L929 cells express this surface antigen. (Connor and Huang, supra:and Huang et al., supra).

We prepared DTA-containing pH-sensitive immunoliposomes composed ofphosphatidylethanolamine (PE)/oleic acid (OA) (8:2 molar ratio) (Connor,Yatkin and Huang, supra) and pH-insensitive immunoliposomes composed ofphosphatidylcholine (PC) using this method. We also preparedDTA-containing liposomes without antibody and empty pH-sensitiveimmunoliposomes of the same lipid composition.

The toxicity of the liposome preparations to L929 cells is shown inFIG. 1. pH-insensitive PC liposomes containing DTA did not inhibitprotein synthesis in the concentration range used. Toxicity of PCliposomes could not be enhanced by incorporation of antibody into theliposome membrane.

It has been shown previously that PC liposomes do not release theirencapsulated contents into the cytoplasm, since they are delivered tothe lysosomes and degraded. (see, Connor and Huang, supra).DTA-containing pH-sensitive liposomes without antibody were alsonon-toxic. This is probably due to a lack of sufficient binding andinternalization of the liposomes. When DTA-containing pH sensitiveimmunoliposomes were incubated with the cells, a dose-dependentinhibition of protein synthesis was observed. This indicates the releaseof active DTA into the cytoplasm. Empty pH-sensitive immunoliposomes hadno effect on protein synthesis.

To see if endosome/lysosome acidification was required for pH-sensitiveimmunoliposome-mediation release of DTA, we did the cytotoxicityexperiments in the presence of NH₄ Cl and chloriquine. These drugs areweak bases which raise the pH of the endosome/lysosome interior (Ohkuma,S., et al., Proc. Natl. Acad. Sci. USA, 75: 3327-3331 (1978) andHelenius, A., et al., J. Gen. Virol., 58: 47-61 (1982).

Cells which were preincubated with either NH₄ Cl or chloriquine prior toimmunoliposome addition were (Huang et al., Biochem. Biophys. Acta, 716:140-150 (1982)) protected from intoxification byimmunoliposome-encapsulated DTA (FIG. 2). Neither NH₄ Cl nor chloroquinealone had an effect on protein synthesis (Huang et al., supra).Therefore endosome/lysosome acidification appears to be required forpH-sensitive immunoliposome-mediated translocation of DTA into thecytoplasm.

We also investigated the dependence of our delivery system on specificcell-surface binding immunoliposomes. Cells were pre-incubated for onehour in the presence of an excess of free antibody prior toimmunoliposome addition. As seen in FIG. 2, such cells were protectedfrom the toxic effect of DTA. Pre-treatment of cells with emptypH-sensitive immunoliposomes before addition of DTA-containingimmunoliposomes also effectively blocked DTA delivery.

It has been shown that DT-resistant mouse cells are not defective in thebinding and internalization of DT (Keen et al., supra) rather, there isa block in the translocation of DTA from the endosome into thecytoplasm. The observation that the pH-sensitive immunoliposomes areable to bypass the translocation block suggests that the site of DTArelease from the liposomes is at the endosome.

This notion is supported by the observation that NH₄ Cl and chloroquineinhibited the observed cytotoxicity. These drugs (Huang et al., supra)are known to inhibit a variety of cellular events which require theacidification of the endosome, such as the release of the Semliki ForestVirus genome (Helenius, et al., supra and the translocation of DTA inthe DT-sensitive cells. (Sandvig, K., et al., J. Cell . Biol., 87:828-832 (1980)).

It is also consistent with preliminary results in which pH-sensitiveimmunoliposomes were able to mediate the cytoplasmic delivery ofcytosine arabinoside. Cytosine arabinoside is a cytotoxic drug which islysosome-sensitive in that exposure of the drug to lysosomal enzymesleads to its degradation and inactivation, (Rustum, Y. M., et al., J.Eur. J. Clin. Oncol., 17: 809-817 (1981).

It is therefore suggested that the pH-sensitive immunoliposomes releasetheir contents into the cytoplasm from the endosomes and the releasestep depends on the acidification of the organelle. The release of theliposome contents is probably the result of liposome-endosome fusion,because it has been shown that pH-sensitive liposomes becomefusion-competent at pH 5-6.5 (Duzgunes, N., et al., Biochem., 24:3091-3098 (1985) and Huang et al., supra) which is the range of theendosome pH (Maxfield, F. R., J. Cell Biol., 95: 676-681 (1982)).

Another possible mechanism of release is that pH-sensitiveimmunoliposomes become leaky and release DTA into the endosome interior.DTA then translocates itself into the cytoplasm as free toxin. This isnot likely since it has been shown that DTA alone cannot cross lipidmembranes in the absence of the B fragment of DT, (Donovan, J. J., etal., J. Biol. Chem., 260: 8817-8823 (1985)).

Another alternative is that pH-sensitive immunoliposomes induce endosomerupture, thereby releasing DTA into the cytoplasm. While the lastmechanism cannot be distinguished from liposome-endosome fusion at thepresent time, the system introduced in this work is more effective forcytoplasmic delivery of DTA than either DTA-antibody or DTA-hormoneconjugates, (Esworthy, R. S., et al., J. Biol. Chem., 259: 11496-11504(1984) and Cawley, D. B., et al., Cell. 22: 563-570 (1980)).

Presumably, the lack of toxicity of these conjugates results fromineffective translocation of DTA into the cytosol since significanttoxicity was obtained only in the presence of added diphtheria Bfragment. (Esworthy and Neville, supra). In contrast, our systemdelivers DTA effectively in the absence of B fragment.

It is clear that pH-sensitive immunoliposomes may be useful for thetargeted, cytoplasmic delivery of other biologically activemacromolecules such as antibodies, enzymes and DNA.

The present invention will be further illustrated with reference to thefollowing examples which aid in the understanding of the presentinvention, but which are not to be construed as limitations thereof. Allpercentages reported herein, unless otherwise specified, are percent byweight. All temperatures are expressed in degrees Celsius.

EXAMPLE 1

Liposomes were prepared by drying appropriate lipids under N₂ gasfollowed by vacuum desiccation. The lipid film was resuspended inphosphate-buffered saline (PBS) and sonicated to form small unilamellarvesicles (SUV). For PE/OA liposomes and immunoliposomes pH was adjustedto 8.0-8.5 by using 0.1N NaOH.

EXAMPLE 2

A 0.2 mg/ml solution of DTA in PBS was added to the SUV of Example 1 andthe mixture was frozen and lyophilized The freeze-dried preparation wasrehydrated with 1/10 of the original SUV volume of either palmiticacid-derivatized antibody (Huang, et al., supra) in 0.15% deoxycholate,PBS, pH 8.0 (for immunoliposomes) or PBS pH 8.0 (for liposomes).

The volume was brought up to 1 ml with PBS and the mixture was extrudedthrough a 0.2 micron filter (Nucleopore). Unencapsulated DTA wasseparated from liposomes by passage over a Sephadex G-200 column(Pharmacia). The average trapping efficiency for the liposomepreparations used was 10% of the available DTA. Antibody incorporatedinto liposome membranes ranged from 38-50% in different experiments. Theimmunoliposomes used in this study contained 0.8 micro-moles DTA and 4micro-moles palmitoyl antibody per 16.5 micro-moles of total lipid.

EXAMPLE 3

Mouse L929 cells (k haplotype) were seeded (10-3 cells/well) into 96well disposable plates (Corning) the day before the experiment. Themedium (McCoy's 5A, supplemented with 10% fetal calf serum) was thenremoved and fresh medium containing immunoliposomes, liposomes or freetoxin was added.

After 3 hours at 37° C. the medium was removed and the cells were washedand fresh medium was added. After 18 hours the medium was replaced withleucine-free McCoy's medium. 2 micro-Ci per well of [³ H]-leucine wasadded and incubation was continued for an additional 6 hr. The cellswere harvested and processed for scintillation counting as described byEsworthy et al., supra.

Results are expressed as the percentage of the incorporation of [³H]-leucine into TCA insoluble material in the untreated controls.Control cells incorporated between 4,000 to 18,000 cpm in differentexperiments. All experiments were done in quadruplicate. Error bars wereincluded only for immunoliposomes containing DTA for the sake ofclarity. The magnitude of error for the other treatments did not differsignificantly from that shown for DTA-containing immunoliposomes.

One hour prior to immunoliposome addition 50 micro-M NH₄ Cl, 50 micro-Mchloroquine, free anti-H-2Kk antibody (10-fold excess) or emptyimmunoliposomes (5-fold excess) were added. Cells were incubated withDTA-containing immunoliposomes, washed, labeled with ³ HI-leucine andtreated as described above.

The present invention has been described in detail, including thepreferred embodiments thereof. However, it will be appreciated thatthose skilled in the art, upon consideration of the present disclosure,may make modifications and/or improvements on this invention and stillbe within the scope and spirit of this invention as set forth in thefollowing claims.

What is claimed is:
 1. Target specific immunoliposomes consistingessentially of antibody coated, pH-sensitive liposomes of 8:2 molarratio of phosphatidylethanolamine and oleic acid, said liposomescontaining at least one entrapped cytotoxic reagent.
 2. The targetspecific immunoliposomes of claim 1, wherein the entrapped cytotoxicreagent is fragment A of the diphtheria toxin.
 3. The target specificimmunoliposomes of claim 1, wherein the antibody coating includes a longchain (C₁₂ -C₂₄) fatty acid segment.
 4. The target specificimmunoliposomes of claim 3, wherein the fatty acid segment is derivedfrom palmitic acid.
 5. The target specific immunoliposomes of claim 1,wherein the antibody is a monoclonal antibody.
 6. The target specificimmunoliposomes of claim 1, wherein the antibody a specific antigen onthe surface of target cells.
 7. The target specific immunoliposomes ofclaim 6, wherein the specific antigen recognized by the antibody is themajor histocompatability antigen H-2K.
 8. A method of deliveringcytotoxic reagents to cells comprising the steps of:(a) preparingpH-sensitive immunoliposomes from phosphatidylethanolamine and oleicacid in a molar ratio of (8:2) and a fatty acid derivatized antibody;(b) entrapping an effective amount of a cytotoxic reagent in thepH-sensitive immunoliposome; and (c) administering said immunoliposomescontaining the cytotoxic reagent to cells expressing an antigen which isrecognized by said antibody.
 9. The method of claim 8, wherein thecytotoxic reagent is an entrapped fragment A of the diphtheria toxin.10. The method of claim 8, wherein the antibody coating includes a longchain (C₁₂ -C₂₄) fatty acid segment.
 11. The method of claim 10, whereinthe fatty acid segment is derived from palmitic acid.
 12. The method ofclaim 8, wherein the antibody is a monoclonal antibody.
 13. The methodof claim 8, wherein the antibody recognizes a specific antigen on thesurface of a target cell.
 14. The method of claim 13, wherein thespecific antigen recognized by the antibody is the majorhistocompatability antigen H-2K.